Nucleotide sequences which code for the menE gene

ABSTRACT

The invention relates to an isolated polynucleotide comprising a polynucleotide sequence chosen from the group consisting of  
     a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,  
     b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2,  
     c) polynucleotide which is complementary to the polynucleotides of a) or b), and  
     d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c),  
     and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the menE gene is present in attenuated form, and the use of polynucleotides which comprise the sequences according to the invention as hybridization probes.

[0001] The invention provides nucleotide sequences from coryneformbacteria which code for the menE gene and a process for the fermentativepreparation of amino acids using bacteria in which the menE gene isattenuated.

[0002] 1. Prior Art

[0003] L-Amino acids, in particular L-lysine, are used in human medicineand in the pharmaceuticals industry, in the foodstuffs industry and veryparticularly in animal nutrition.

[0004] It is known that amino acids are prepared by fermentation fromstrains of coryneform bacteria, in particular Corynebacteriumglutamicum. Because of their great importance, work is constantly beingundertaken to improve the preparation processes. Improvements to theprocess can relate to fermentation measures, such as, for example,stirring and supply of oxygen, or the composition of the nutrient media,such as, for example, the sugar concentration during the fermentation,or the working up to the product form by, for example, ion exchangechromatography, or the intrinsic output properties of the microorganismitself.

[0005] Methods of mutagenesis, selection and mutant selection are usedto improve the output properties of these microorganisms. Strains whichare resistant to antimetabolites or are auxotrophic for metabolites ofregulatory importance and which produce amino acids are obtained in thismanner.

[0006] Methods of the recombinant DNA technique have also been employedfor some years for improving the strain of Corynebacterium strains whichproduce L-amino acid, by amplifying individual amino acid biosynthesisgenes and investigating the effect on the amino acid production.

[0007] 2. Object of the Invention

[0008] The inventors had the object of providing new measures forimproved fermentative preparation of amino acids.

DESCRIPTION OF THE INVENTION

[0009] Where L-amino acids or amino acids are mentioned in thefollowing, this means one or more amino acids, including their salts,chosen from the group consisting of L-asparagine, L-threonine, L-serine,L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,L-lysine, L-tryptophan and L-arginine. L-Lysine is particularlypreferred.

[0010] When L-lysine or lysine are mentioned in the following, not onlythe bases but also the salts, such as e.g. lysine monohydrochloride orlysine sulfate, are meant by this.

[0011] The invention provides an isolated polynucleotide from coryneformbacteria, comprising a polynucleotide sequence which codes for the menEgene, chosen from the group consisting of

[0012] a) polynucleotide which is identical to the extent of at least70% to a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequence of SEQ ID No. 2,

[0013] b) polynucleotide which codes for a polypeptide which comprisesan amino acid sequence which is identical to the extent of at least 70%to the amino acid sequence of SEQ ID No. 2,

[0014] c) polynucleotide which is complementary to the polynucleotidesof a) or b), and

[0015] d) polynucleotide comprising at least 15 successive nucleotidesof the polynucleotide sequence of a), b) or c),

[0016] the polypeptide preferably having the activity ofO-succinylbenzoic acid CoA ligase.

[0017] The invention also provides the abovementioned polynucleotide,this preferably being a DNA which is capable of replication, comprising:

[0018] (i) the nucleotide sequence, shown in SEQ ID No.1, or

[0019] (ii) at least one sequence which corresponds to sequence (i)within the range of the degeneration of the genetic code, or

[0020] (iii) at least one sequence which hybridizes with the sequencescomplementary to sequences (i) or (ii), and optionally

[0021] (iv) sense mutations of neutral function in (i).

[0022] The invention also provides:

[0023] a polynucleotide, in particular DNA, which is capable ofreplication and comprises the nucleotide sequence as shown in SEQ IDNo.1;

[0024] a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequence as shown in SEQ ID No. 2;

[0025] a vector containing parts of the polynucleotide according to theinvention, but at least 15 successive nucleotides of the sequenceclaimed,

[0026] and coryneform bacteria in which the menE gene is attenuated, inparticular by an insertion or deletion.

[0027] The invention also provides polynucleotides which substantiallycomprise a polynucleotide sequence, which are obtainable by screening bymeans of hybridization of a corresponding gene library of a coryneformbacterium, which comprises the complete gene or parts thereof, with aprobe which comprises the sequence of the polynucleotide according tothe invention according to SEQ ID No.1 or a fragment thereof, andisolation of the polynucleotide sequence mentioned.

[0028] Polynucleotides which comprise the sequences according to theinvention are suitable as hybridization probes for RNA, cDNA and DNA, inorder to isolate, in the full length, nucleic acids or polynucleotidesor genes which code for O-succinylbenzoic acid CoA ligase or to isolatethose nucleic acids or polynucleotides or genes which have a highsimilarity with the sequence the menE gene.

[0029] Polynucleotides which comprise the sequences according to theinvention are furthermore suitable as primers with the aid of which DNAof genes which code for O-succinylbenzoic acid CoA ligase can beprepared by the polymerase chain reaction (PCR).

[0030] Such oligonucleotides which serve as probes or primers compriseat least 30, preferably at least 20, very particularly preferably atleast 15 successive nucleotides. Oligonucleotides which have a length ofat least 40 or 50 nucleotides are also suitable.

[0031] “Isolated” means separated out of its natural environment.

[0032] “Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

[0033] The polynucleotides according to the invention include apolynucleotide according to SEQ ID No. 1 or a fragment preparedtherefrom and also those which are at least 70%, preferably at least 80%and in particular at least 90% to 95% identical to the polynucleotideaccording to SEQ ID No. 1 or a fragment prepared therefrom.

[0034] “Polypeptides” are understood as meaning peptides or proteinswhich comprise two or more amino acids bonded via peptide bonds.

[0035] The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2, in particular those with the biologicalactivity of O-succinylbenzoic acid CoA ligase, and also those which areat least 70%, preferably at least 80% and in particular at least 90% to95% identical to the polypeptide according to SEQ ID No. 2 and have theactivity mentioned.

[0036] The invention furthermore relates to a process for thefermentative preparation of amino acids chosen from the group consistingof L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine,L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine,L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan andL-arginine using coryneform bacteria which in particular already produceamino acids and in which the nucleotide sequences which code for themenE gene are attenuated, in particular eliminated or expressed at a lowlevel.

[0037] The term “attenuation” in this connection describes the reductionor elimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or using a gene or allele whichcodes for a corresponding enzyme with a low activity or inactivates thecorresponding gene or enzyme (protein), and optionally combining thesemeasures.

[0038] The microorganisms to which the present invention relates canprepare amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, starch, cellulose or from glycerol and ethanol. They can berepresentatives of coryneform bacteria, in particular of the genusCorynebacterium. Of the genus Corynebacterium, there may be mentioned inparticular the species Corynebacterium glutamicum, which is known amongexperts for its ability to produce L-amino acids.

[0039] Suitable strains of the genus Corynebacterium, in particular ofthe species Corynebacterium glutamicum (C. glutamicum), are inparticular the known wild-type strains

[0040]Corynebacterium glutamicum ATCC13032

[0041]Corynebacterium acetoglutamicum ATCC15806

[0042]Corynebacterium acetoacidophilum ATCC13870

[0043]Corynebacterium melassecola ATCC17965

[0044]Corynebacterium thermoaminogenes FERM BP-1539

[0045]Brevibacterium flavum ATCC14067

[0046]Brevibacterium lactofermentum ATCC13869 and

[0047]Brevibacterium divaricatum ATCC14020 and L-amino acid-producingmutants or strains prepared therefrom.

[0048] The new menE gene from C. glutamicum which codes for the enzymeO-succinylbenzoic acid CoA ligase (EC 6.2.1.26) has been isolated.

[0049] To isolate the menE gene or also other genes of C. glutamicum, agene library of this microorganism is first set up in Escherichia coli(E. coli). The setting up of gene libraries is described in generallyknown textbooks and handbooks. The textbook by Winnacker: Gene undKlone, Eine Einführung in die Gentechnologie (Verlag Chemie, Weinheim,Germany, 1990), or the handbook by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may bementioned as an example. A well-known gene library is that of the E.coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50,495 -508 (1987)). Bathe et al. (Molecular and General Genetics,252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032,which was set up with the aid of the cosmid vector SuperCos I (Wahl etal., 1987, Proceedings of the National Academy of Sciences USA,84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988,Nucleic Acids Research 16:1563-1575).

[0050] Börmann et al. (Molecular Microbiology 6(3), 317-326)) (1992)) inturn describe a gene library of C. glutamicum ATCC13032 using the cosmidpHC79 (Hohn and Collins, 1980, Gene 11, 291-298).

[0051] To prepare a gene library of C. glutamicum in E. coli it is alsopossible to use plasmids such as pBR322 (Bolivar, 1979, Life Sciences,25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitablehosts are, in particular, those E. coli strains which are restriction-and recombination-defective, such as, for example, the strain DH5αmcr,which has been described by Grant et al. (Proceedings of the NationalAcademy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragmentscloned with the aid of cosmids or other λ vectors can then in turn besubcloned and subsequently sequenced in the usual vectors which aresuitable for DNA sequencing, such as is described e. g. by Sanger et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica, 74:5463-5467, 1977).

[0052] The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232(1986)), that of Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program of Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

[0053] The new DNA sequence of C. glutamicum which codes for the menEgene and which, as SEQ ID No. 1, is a constituent of the presentinvention has been found. The amino acid sequence of the correspondingprotein has furthermore been derived from the present DNA sequence bythe methods described above. The resulting amino acid sequence of themenE gene product is shown in SEQ ID No. 2.

[0054] Coding DNA sequences which result from SEQ ID No. 1 by thedegeneracy of the genetic code are also a constituent of the invention.In the same way, DNA sequences which hybridize with SEQ ID No. 1 orparts of SEQ ID No. 1 are a constituent of the invention. Conservativeamino acid exchanges, such as e.g. exchange of glycine for alanine or ofaspartic acid for glutamic acid in proteins, are furthermore known amongexperts as “sense mutations” which do not lead to a fundamental changein the activity of the protein, i.e. are of neutral function. It isfurthermore known that changes on the N and/or C terminus of a proteincannot substantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), inO'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (ProteinSciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology6:1321-1325 (1988)) and in known textbooks of genetics and molecularbiology. Amino acid sequences which result in a corresponding mannerfrom SEQ ID No. 2 are also a constituent of the invention.

[0055] In the same way, DNA sequences which hybridize with SEQ ID No. 1or parts of SEQ ID No. 1 are a constituent of the invention. Finally,DNA sequences which are prepared by the polymerase chain reaction (PCR)using primers which result from SEQ ID No. 1 are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

[0056] Instructions for identifying DNA sequences by means ofhybridization can be found by the expert, inter alia, in the handbook“The DIG System Users Guide for Filter Hybridization” from BoehringerMannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al.(International Journal of Systematic Bacteriology 41: 255-260 (1991)).The hybridization takes place under stringent conditions, that is to sayonly hybrids in which the probe and target sequence, i. e. thepolynucleotides treated with the probe, are at least 70% identical areformed. It is known that the stringency of the hybridization, includingthe washing steps, is influenced or determined by varying the buffercomposition, the temperature and the salt concentration. Thehybridization reaction is preferably carried out under a relatively lowstringency compared with the washing steps (Hybaid Hybridisation Guide,Hybaid Limited, Teddington, UK, 1996).

[0057] A 5x SSC buffer at a temperature of approx. 50° C.- 68° C., forexample, can be employed for the hybridization reaction. Probes can alsohybridize here with polynucleotides which are less than 70% identical tothe sequence of the probe. Such hybrids are less stable and are removedby washing under stringent conditions. This can be achieved, forexample, by lowering the salt concentration to 2×SSC and optionallysubsequently 0.5×SSC (The DIG System User's Guide for FilterHybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) atemperature of approx. 50° C.-68° C. being established. It is optionallypossible to lower the salt concentration to 0.1×SSC. Polynucleotidefragments which are, for example, at least 70% or at least 80% or atleast 90% to 95% identical to the sequence of the probe employed can beisolated by increasing the hybridization temperature stepwise from 50°C. to 68° C. in steps of approx. 1-2° C. Further instructions onhybridization are obtainable on the market in the form of so-called kits(e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany,Catalogue No. 1603558).

[0058] Instructions for amplification of DNA sequences with the aid ofthe polymerase chain reaction (PCR) can be found by the expert, interalia, in the handbook by Gait: Oligonukleotide [sic] synthesis: APractical Approach (IRL Press, Oxford, UK, 1984) and in Newton andGraham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

[0059] It has been found that coryneform bacteria produce amino acids inan improved manner after attenuation of the menE gene.

[0060] To achieve an attenuation, either the expression of the menE geneor the catalytic properties of the enzyme protein can be reduced oreliminated. The two measures can optionally be combined.

[0061] The reduction in gene expression can take place by suitableculturing or by genetic modification (mutation) of the signal structuresof gene expression. Signal structures of gene expression are, forexample, repressor genes, activator genes, operators, promoters,attenuators, ribosome binding sites, the start codon and terminators.The expert can find information on this e.g. in the patent applicationWO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949(1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548(1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191(1998)), in Patek et al. (Microbiology 142: 1297 (1996)), Vasicova etal. (Journal of Bacteriology 181: 6188 (1999)) and in known textbooks ofgenetics and molecular biology, such as e.g. the textbook by Knippers(“Molekulare Genetik”, 6th edition, Georg Thieme Verlag, Stuttgart,Germany, 1995) or that by Winnacker (“Gene und Klone”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990).

[0062] Mutations which lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art; exampleswhich may be mentioned are the works by Qiu and Goodman (Journal ofBiological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (BioscienceBiotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“DieThreonindehydratase aus Corynebacterium glutamicum: Aufhebung derallosterischen Regulation und Struktur des Enzyms”, Reports from theJülich Research Centre, Jül-2906, ISSN09442952, Jülich, Germany, 1994).Summarizing descriptions can be found in known textbooks of genetics andmolecular biology, such as e.g. that by Hagemann (“Allgemeine Genetik”,Gustav Fischer Verlag, Stuttgart, 1986).

[0063] Possible mutations are transitions, transversions, insertions anddeletions. Depending on the effect of the amino acid exchange on theenzyme activity, “missense mutations” or “nonsense mutations” arereferred to. Insertions or deletions of at least one base pair (bp) in agene lead to frame shift mutations, as a consequence of which incorrectamino acids are incorporated or translation is interrupted prematurely.Deletions of several codons typically lead to a complete loss of theenzyme activity. Instructions on generation of such mutations are priorart and can be found in known textbooks of genetics and molecularbiology, such as e.g. the textbook by Knippers (“Molekulare Genetik”,6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that byWinnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim, Germany,1990) or that by Hagemann (“Allgemeine Genetik”, Gustav Fischer Verlag,Stuttgart, 1986).

[0064] A common method of mutating genes of C. glutamicum is the methodof “gene disruption” and “gene replacement” described by Schwarzer andPüthler (Bio/Technology 9, 84-87 (1991)).

[0065] In the method of gene disruption a central part of the codingregion of the gene of interest is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), pK18mobsacB or pK19mobsacB (Jäger et al., Journal ofBacteriology 174: 5462-65 (1992)), pGEM-T (Promega corporation, Madison,Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen,Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology173:4510-4516). The plasmid vector which contains the central part ofthe coding region of the gene is then transferred into the desiredstrain of C. glutamicum by conjugation or transformation. The method ofconjugation is described, for example, by Schäfer et al. (Applied andEnvironmental Microbiology 60, 756-759 (1994)). Methods fortransformation are described, for example, by Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)). After homologousrecombination by means of a “cross-over” event, the coding region of thegene in question is interrupted by the vector sequence and twoincomplete alleles are obtained, one lacking the 3′ end and one lackingthe 5′ end. This method has been used, for example, by Fitzpatrick etal. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) toeliminate the recA gene of C. glutamicum.

[0066] In the method of “gene replacement”, a mutation, such as e.g. adeletion, insertion or base exchange, is established in vitro in thegene of interest. The allele prepared is in turn cloned in a vectorwhich is not replicative for C. glutamicum and this is then transferredinto the desired host of C. glutamicum by transformation or conjugation.After homologous recombination by means of a first “cross-over” eventwhich effects integration and a suitable second “cross-over” event whicheffects excision in the target gene or in the target sequence, theincorporation of the mutation or of the allele is achieved. This methodwas used, for example, by Peters-Wendisch et al. (Microbiology 144,915 - 927 (1998)) to eliminate the pyc gene of C. glutamicum by adeletion.

[0067] A deletion, insertion or a base exchange can be incorporated intothe menE gene in this manner.

[0068] In addition, it may be advantageous for the production of L-aminoacids to enhance, in particular over-express, one or more enzymes of theparticular biosynthesis pathway, of glycolysis, of anaplerosis, of thecitric acid cycle, of the pentose phosphate cycle, of amino acid exportand optionally regulatory proteins, in addition to the attenuation ofthe menE gene.

[0069] The term “enhancement” in this connection describes the increasein the intracellular activity of one or more enzymes (proteins) in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene or allele which codes for a correspondingenzyme (protein) having a high activity, and optionally combining thesemeasures.

[0070] Thus, for the preparation of L-amino acids, in addition to theattenuation of the menE gene at the same time one or more of the geneschosen from the group consisting of

[0071] the dapA gene which codes for dihydrodipicolinate synthase (EP-B0 197 335),

[0072] the gap gene which codes for glyceraldehyde 3-phosphatedehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

[0073] the tpi gene which codes for triose phosphate isomerase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0074] the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0075] the zwf gene which codes for glucose 6-phosphate dehydrogenase(JP-A-09224661),

[0076] the pyc gene which codes for pyruvate carboxylase (DE-A-198 31609),

[0077] the mqo gene which codes for malate-quinone oxidoreductase(Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),

[0078] the lysC gene which codes for a feed-back resistant aspartatekinase (Accession No.P26512; EP-B-0387527; EP-A-0699759; WO 00/63388),

[0079] the lysE gene which codes for lysine export (DE-A-195 48 222),

[0080] the hom gene which codes for homoserine dehydrogenase (EP-A0131171),

[0081] the ilvA gene which codes for threonine dehydratase (Möckel etal., Journal of Bacteriology (1992) 8065-8072)) or the ilvA(Fbr) allelewhich codes for a “feed back resistant” threonine dehydratase (Möckel etal., (1994) Molecular Microbiology 13: 833-842),

[0082] the ilvBN gene which codes for acetohydroxy-acid synthase (EP-B0356739),

[0083] the ilvD gene which codes for dihydroxy-acid dehydratase (Sahmand Eggeling (1999) Applied and Environmental Microbiology 65:1973-1979),

[0084] the zwal gene which codes for the Zwal protein (DE: 19959328.0,DSM 13115) can be enhanced, in particular over-expressed.

[0085] It may furthermore be advantageous for the production of aminoacids, in addition to the attenuation of the menE gene, at the same timefor one or more of the genes chosen from the group consisting of

[0086] the pck gene which codes for phosphoenol pyruvate carboxykinase(DE 199 50 409.1, DSM 13047),

[0087] the pgi gene which codes for glucose 6-phosphate isomerase (US09/396,478, DSM 12969),

[0088] the poxB gene which codes for pyruvate oxidase (DE:1995 1975.7,DSM 13114),

[0089] the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2,DSM 13113) to be attenuated, in particular for the expression thereof tobe reduced.

[0090] In addition to the attenuation of the menE gene it mayfurthermore be advantageous for the production of amino acids toeliminate undesirable side reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

[0091] The invention also provides the microorganisms prepared accordingto the invention, and these can be cultured continuously ordiscontinuously in the batch process (batch culture) or in the fed batch(feed process) or repeated fed batch process (repetitive feed process)for the purpose of production of L-amino acids. A summary of knownculture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (GustavFischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

[0092] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

[0093] Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, starch and cellulose, oils and fats, suchas, for example, soya oil, sunflower oil, groundnut oil and coconut fat,fatty acids, such as, for example, palmitic acid, stearic acid andlinoleic acid, alcohols, such as, for example, glycerol and ethanol, andorganic acids, such as, for example, acetic acid, can be used as thesource of carbon. These substances can be used individually or as amixture.

[0094] Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture.

[0095] Phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts can beused as the source of phosphorus. The culture medium must furthermorecomprise salts of metals, such as, for example, magnesium sulfate oriron sulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids and vitamins, can be employed inaddition to the abovementioned substances. Suitable precursors canmoreover be added to the culture medium. The starting substancesmentioned can be added to the culture in the form of a single batch, orcan be fed in during the culture in a suitable manner.

[0096] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture. Antifoams, such as, for example, fatty acid polyglycolesters, can be employed to control the development of foam. Suitablesubstances having a selective action, such as, for example, antibiotics,can be added to the medium to maintain the stability of plasmids. Tomaintain aerobic conditions, oxygen or oxygen-containing gas mixtures,such as, for example, air, are introduced into the culture. Thetemperature of the culture is usually 20° C. to 45° C., and preferably25° C. to 40° C. Culturing is continued until a maximum of the desiredproduct has formed. This target is usually reached within 10 hours to160 hours.

[0097] Methods for the determination of L-amino acids are known from theprior art. The analysis can thus be carried out, for example, asdescribed by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) byanion exchange chromatography with subsequent ninhydrin derivatization,or it can be carried out by reversed phase HPLC, for example asdescribed by Lindroth et al. (Analytical Chemistry (1979) 51:1167-1174).

[0098] The process according to the invention is used for fermentativepreparation of amino acids.

[0099] The following microorganism was deposited on 26.02.2001 as a pureculture at the Deutsche Sammlung für Mikrorganismen [sic] undZellkulturen (DSMZ=German Collection of Microorganisms and CellCultures, Braunschweig, Germany) in accordance with the Budapest Treaty:

[0100]Escherichia coli Top10/pCR2.1menEint as DSM 14080.

[0101] The present invention is explained in more detail in thefollowing with the aid of embodiment examples.

[0102] The isolation of plasmid DNA from Escherichia coli and alltechniques of restriction, Klenow and alkaline phosphatase treatmentwere carried out by the method of Sambrook et al. (Molecular Cloning. ALaboratory Manual, 1989, Cold Spring Harbour Laboratory Press, ColdSpring Harbor, N.Y., USA). Methods for transformation of Escherichiacoli are also described in this handbook.

[0103] The composition of the usual nutrient media, such as LB or TYmedium, can also be found in the handbook by Sambrook et al.

Example 1

[0104] Preparation of a genomic cosmid gene library from C. glutamicumATCC 13032

[0105] Chromosomal DNA from C. glutamicum ATCC 13032 was isolated asdescribed by Tauch et al. (1995, Plasmid 33:168-179) and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Code no. 27-0913-02). The DNAfragments were dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP, Codeno. 1758250). The DNA of the cosmid vector SuperCosl (Wahl et al.(1987), Proceedings of the National Academy of Sciences, USA84:2160-2164), obtained from Stratagene (La Jolla, USA, ProductDescription SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,Product Description XbaI, Code no. 27-0948-02) and likewisedephosphorylated with shrimp alkaline phosphatase.

[0106] The cosmid DNA was then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04). The cosmid DNA treated in this manner was mixed withthe treated ATCC13032 DNA and the batch was treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no.27-0870-04). The ligation mixture was then packedin phages with the aid of Gigapack II XL Packing Extract (Stratagene, LaJolla, USA, Product Description Gigapack II XL Packing Extract, Code no.200217).

[0107] For infection of the E. coli strain NM554 (Raleigh et al. 1988,Nucleic Acid Res. 16:1563-1575) the cells were taken up in 10 mM MgSO₄and mixed with an aliquot of the phage suspension. The infection andtitering of the cosmid library were carried out as described by Sambrooket al. (1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor), the cells being plated out on LB agar (Lennox, 1955, Virology,1:190) +100 ug/ml ampicillin. After incubation overnight at 37° C.,recombinant individual clones were selected.

Example 2

[0108] Isolation and sequencing of the menE gene

[0109] The cosmid DNA of an individual colony was isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's instructions and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNAfragments were dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP,Product No. 1758250). After separation by gel electrophoresis, thecosmid fragments in the size range of 1500 to 2000 bp were isolated withthe QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden,Germany).

[0110] The DNA of the sequencing vector pZero-1, obtained fromInvitrogen (Groningen, The Netherlands, Product Description ZeroBackground Cloning Kit, Product No. K2500-01) was cleaved with therestriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, ProductDescription BamHI, Product No. 27-0868-04). The ligation of the cosmidfragments in the sequencing vector pZero-1 was carried out as describedby Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor), the DNA mixture being incubated overnight with T4 ligase(Pharmacia Biotech, Freiburg, Germany). This ligation mixture was thenelectroporated (Tauch et al. 1994, FEMS Microbiol. Letters, 123:343-7)into the E. coli strain DH5αmcr (Grant, 1990, Proceedings of theNational Academy of Sciences, U.S.A., 87:4645-4649). Letters, 123:343-7)and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 μg/mlzeocin.

[0111] The plasmid preparation of the recombinant clones was carried outwith Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Thesequencing was carried out by the dideoxy chain termination method ofSanger et al. (1977, Proceedings of the National Academies of Sciences,U.S.A., 74:5463-5467) with modifications according to Zimmermann et al.(1990, Nucleic Acids Research, 18:1067). The “RR dRhodamin TerminatorCycle Sequencing Kit” from PE Applied Biosystems (Product No. 403044,Weiterstadt, Germany) was used. The separation by gel electrophoresisand analysis of the sequencing reaction were carried out in a“Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (Product No.A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencerfrom PE Applied Biosystems (Weiterstadt, Germany).

[0112] The raw sequence data obtained were then processed using theStaden program package (1986, Nucleic Acids Research, 14:217-231)version 97-0. The individual sequences of the pZero1 derivatives wereassembled to a continuous contig. The computer-assisted coding regionanalysis [sic] were prepared with the XNIP program (Staden, 1986,Nucleic Acids Research, 14:217-231). Further analyses were carried outwith the “BLAST search program” (Altschul et al., 1997, Nucleic AcidsResearch, 25:3389-3402) against the non-redundant databank of the“National Center for Biotechnology Information” (NCBI, Bethesda, Md.,USA).

[0113] The resulting nucleotide sequence is shown in SEQ ID No. 1.Analysis of the nucleotide sequence showed an open reading frame of 1131bp, which was called the menE gene. The menE gene codes for apolypeptide of 376 amino acids.

Example 3

[0114] Preparation of an integration vector for integration mutagenesisof the menE gene

[0115] From the strain ATCC 13032, chromosomal DNA was isolated by themethod of Eikmanns et al. (Microbiology 140: 1817 -1828 (1994)). On thebasis of the sequence of the menE gene known for C. glutamicum fromexample 2, the following oligonucleotides were chosen for the polymerasechain reaction (see SEQ ID No. 3 and SEQ ID No. 4):

[0116] menE-int1: 5′ CTC ACT CCG TTG AAT TTG G 3′

[0117] menE-int2: 5′ CAG GTG CAT TTC TGT AGC C 3′

[0118] The primers shown were synthesized by MWG Biotech (Ebersberg,Germany) and the PCR reaction was carried out by the standard PCR methodof Innis et al. (PCR protocols. A guide to methods and applications,1990, Academic Press) with the Taq-polymerase from Boehringer Mannheim(Germany, Product Description Taq DNA polymerase, Product No. 1 146165). With the aid of the polymerase chain reaction, the primers allowamplification of an internal fragment of the menE gene 520 bp in size.The product amplified in this way was tested electrophoretically in a0.8% agarose gel.

[0119] The amplified DNA fragment was ligated with the TOPO TA CloningKit from Invitrogen Corporation (Carlsbad, Calif., USA; Catalogue NumberK4500-01) in the vector pCR2.1-TOPO (Mead at al. (1991) Bio/Technology9:657-663).

[0120] The E. coli strain TOP10 was then electroporated with theligation batch (Hanahan, In: DNA cloning. A practical approach. Vol.I.IRL-Press, Oxford, Washington D.C., USA, 1985). Selection ofplasmid-carrying cells was carried out by plating out the transformationbatch on LB Agar (Sambrook et al., Molecular cloning: a laboratorymanual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), which had been supplemented with 50 mg/l kanamycin. PlasmidDNA was isolated from a transformant with the aid of the QIAprep SpinMiniprep Kit from Qiagen and checked by restriction with the restrictionenzyme EcoRI and subsequent agarose gel electrophoresis (0.8%). Theplasmid was called pCR2.1menEint and is shown in FIG. 1.

Example 4

[0121] Integration mutagenesis of the menE gene in the strain DSM 5715

[0122] The vector pCR2.1menEint mentioned in example 3 waselectroporated by the electroporation method of Tauch et al.(FEMSMicrobiological Letters, 123:343-347 (1994)) in Corynebacteriumglutamicum DSM 5715. The strain DSM 5715 is an AEC-resistant lysineproducer. The vector pCR2.1menEint cannot replicate independently inDSM5715 and is retained in the cell only if it has integrated into thechromosome of DSM 5715. Selection of clones with pCR2.1menEintintegrated into the chromosome was carried out by plating out theelectroporation batch on LB agar (Sambrook et al., Molecular cloning: alaboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.), which had been supplemented with 15 mg/lkanamycin.

[0123] For detection of the integration, the menEint fragment waslabelled with the Dig hybridization kit from Boehringer by the method of“The DIG System Users Guide for Filter Hybridization” of BoehringerMannheim GmbH (Mannheim, Germany, 1993). Chromosomal DNA of a potentialintegrant was isolated by the method of Eikmanns et al. (Microbiology140: 1817 - 1828 (1994)) and in each case cleaved with the restrictionenzymes EcoRI and PstI. The fragments formed were separated by means ofagarose gel electrophoresis and hybridized at 68° C. with the Dighybrization [sic] kit from Boehringer. The plasmid pCR2.1menEintmentioned in example 3 had been inserted into the chromosome of DSM5715within the chromosomal menE gene. The strain was calledDSM5715::pCR2.1menEint.

Example 5

[0124] Preparation of lysine

[0125] The C. glutamicum strain DSM5715::pCR2.1menEint obtained inexample 4 was cultured in a nutrient medium suitable for the productionof lysine and the lysine content in the culture supernatant wasdetermined.

[0126] For this, the strain was first incubated on an agar plate withthe corresponding antibiotic (brain-heart agar with kanamycin (25 mg/l)for 24 hours at 33° C. Starting from this agar plate culture, apreculture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium CgIII was used as the medium for the preculture. MediumCg III NaCl 2.5 g/l Bacto-Peptone 10 g/l Bacto-Yeast extract 10 g/lGlucose (autoclaved separately) 2% (w/v) The pH was brought to pH 7.4

[0127] Kanamycin (25 mg/l) was added to this. The preculture wasincubated for 16 hours at 33° C. at 240 rpm on a shaking machine. A mainculture was seeded from this preculture such that the initial OD (660nm) of the main culture was 0.1 OD. Medium MM was used for the mainculture. Medium MM CSL (corn steep liquor) 5 g/l MOPS(morpholinopropanesulfonic acid) 20 g/l Glucose (autoclaved separately)50 g/l Salts: (NH₄)₂SO₄) 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/lCaCl₂ * 2 H₂O 10 mg/l FeSO₄ * 7 H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin(sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2 mg/lLeucine (sterile-filtered) 0.1 g/l CaCO₃ 25 g/l

[0128] The CSL, MOPS and the salt solution are brought to pH 7 withaqueous ammonia and autoclaved. The sterile substrate and vitaminsolutions are then added, and the CaCO₃ autoclaved in the dry state isadded.

[0129] Culturing is carried out in a 10 ml volume in a 100 ml conicalflask with baffles. Kanamycin (25 mg/l) was added. Culturing was carriedout at 33° C. and 80% atmospheric humidity.

[0130] After 72 hours, the OD was determined at a measurement wavelengthof 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). Theamount of lysine formed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivatization with ninhydrin detection. The result ofthe experiment is shown in table 1. TABLE 1 Lysine HCl Strain OD(660)g/l DSM5715 8.2 12.64 DSM5715::pCR2.1menEint 9.2 14.89

[0131] Description of the figure:

[0132]FIG. 1: Map of the plasmid pCR2.1menEint.

[0133] The abbreviations and designations used have the followingmeaning.

[0134] KmR: Kanamycin resistance gene

[0135] EcoRI: Cleavage site of the restriction enzyme EcoRI

[0136] PstI: Cleavage site of the restriction enzyme PstI

[0137] meneint: Internal fragment of the menE gene

[0138] ColE1: Replication origin of the plasmid ColE1

[0139]

1 4 1 1570 DNA Corynebacterium glutamicum CDS (230)..(1357) 1 ttcgttgccatagacatgct cttcgcagca ctgtttgcgc acgtctcctc cggcatcttt 60 gtcaccaacaatggttggga actcaccggc gcaatcggcg ctggcgcgct gcttctcatc 120 gcagttggcgcaggtgcatg gagcatcgac ggggttctgg caaaacgcaa ggcctaaatc 180 tagcgccacaactccgaatt ctgaaccatc ggcactagaa tctcggaat atg aat act 238 Met Asn Thr 1cgc gtc ctc gaa gca cta cct gtt gat ctt gca gat ccc acc gca att 286 ArgVal Leu Glu Ala Leu Pro Val Asp Leu Ala Asp Pro Thr Ala Ile 5 10 15 ctggga gat ctc gag gac gca atc tct ggg aag aaa act ttc ctc ccc 334 Leu GlyAsp Leu Glu Asp Ala Ile Ser Gly Lys Lys Thr Phe Leu Pro 20 25 30 35 atccct gta caa gat aaa acc cgt gca cag ttg ctg cgc gat tct caa 382 Ile ProVal Gln Asp Lys Thr Arg Ala Gln Leu Leu Arg Asp Ser Gln 40 45 50 cga gttggc ctc gcc atc gat cct tcg atc gct ttg gtg atg gcc act 430 Arg Val GlyLeu Ala Ile Asp Pro Ser Ile Ala Leu Val Met Ala Thr 55 60 65 tct ggt tctaca ggt acc ccg aag ggc gct cag ctc act ccg ttg aat 478 Ser Gly Ser ThrGly Thr Pro Lys Gly Ala Gln Leu Thr Pro Leu Asn 70 75 80 ttg gtg agt tccgcc gat gct acg cat cag ttt tta ggt ggc gaa ggc 526 Leu Val Ser Ser AlaAsp Ala Thr His Gln Phe Leu Gly Gly Glu Gly 85 90 95 cag tgg ttg ctt gccatg cca gca cac cac att gca ggc atg cag gtg 574 Gln Trp Leu Leu Ala MetPro Ala His His Ile Ala Gly Met Gln Val 100 105 110 115 ctt ctt cga agcctc att gct gga gtt gag cca cta gct att gat ctc 622 Leu Leu Arg Ser LeuIle Ala Gly Val Glu Pro Leu Ala Ile Asp Leu 120 125 130 agc aca ggt tttcac att gac gct ttc gca ggc gcc gcg gca gaa ctg 670 Ser Thr Gly Phe HisIle Asp Ala Phe Ala Gly Ala Ala Ala Glu Leu 135 140 145 aaa aat acc ggcgac cgc gtc tat aca tcc ttg act cca atg cag tta 718 Lys Asn Thr Gly AspArg Val Tyr Thr Ser Leu Thr Pro Met Gln Leu 150 155 160 ctt aaa gca atggac tcc ttg caa ggc att gaa gcc ctg aaa ctt ttt 766 Leu Lys Ala Met AspSer Leu Gln Gly Ile Glu Ala Leu Lys Leu Phe 165 170 175 gat gtc att cttgtt ggc ggt gct gca ttg tct aag cag gcc cga att 814 Asp Val Ile Leu ValGly Gly Ala Ala Leu Ser Lys Gln Ala Arg Ile 180 185 190 195 tct gcg gagcag cta gac atc aac att gtc acc acc tac ggc tcc tca 862 Ser Ala Glu GlnLeu Asp Ile Asn Ile Val Thr Thr Tyr Gly Ser Ser 200 205 210 gag act tcaggt ggc tgc gtt tat gat ggc aag ccc att ccc ggc gcg 910 Glu Thr Ser GlyGly Cys Val Tyr Asp Gly Lys Pro Ile Pro Gly Ala 215 220 225 aaa gtc cgtatt tcg gat gag cgc att gag ttg ggt ggc ccg atg att 958 Lys Val Arg IleSer Asp Glu Arg Ile Glu Leu Gly Gly Pro Met Ile 230 235 240 gcg cag ggctac aga aat gca cct gaa cat ccg gat ttc gcc aac gag 1006 Ala Gln Gly TyrArg Asn Ala Pro Glu His Pro Asp Phe Ala Asn Glu 245 250 255 ggt tgg tttacc acc tct gat tca ggt gaa ctc cac gac ggg att ctc 1054 Gly Trp Phe ThrThr Ser Asp Ser Gly Glu Leu His Asp Gly Ile Leu 260 265 270 275 acc gtgact ggt cgc gtg gat acc gtc att gat tcc ggt gga ttg aag 1102 Thr Val ThrGly Arg Val Asp Thr Val Ile Asp Ser Gly Gly Leu Lys 280 285 290 ttg caccca gag gta ctg gaa cgt gcc atc gca gat att aaa ggt gtc 1150 Leu His ProGlu Val Leu Glu Arg Ala Ile Ala Asp Ile Lys Gly Val 295 300 305 acc gcggcg tgt gtt gtg ggt att ccc gat ccc cga tta ggc caa gca 1198 Thr Ala AlaCys Val Val Gly Ile Pro Asp Pro Arg Leu Gly Gln Ala 310 315 320 att gtggcc gcg tac tcc gga tcg atc agt ccg tct gaa gtt att gaa 1246 Ile Val AlaAla Tyr Ser Gly Ser Ile Ser Pro Ser Glu Val Ile Glu 325 330 335 ggc ctcgac gat cta cct cgt tgg cag ctt ccc aaa cgg ctg aag cat 1294 Gly Leu AspAsp Leu Pro Arg Trp Gln Leu Pro Lys Arg Leu Lys His 340 345 350 355 ctggaa tct ttg ccc agc att ggt cct gga aaa gct gat cga cgt gct 1342 Leu GluSer Leu Pro Ser Ile Gly Pro Gly Lys Ala Asp Arg Arg Ala 360 365 370 atcgcg aag ctg ttt tagtcttcat tcttgctggc tgcaactagt tttgccacat 1397 Ile AlaLys Leu Phe 375 cttcatcggt gtacactttg gcgatctgct catcatttcc acccatgagggtgttgccaa 1457 caactagtgc tcccacttgg gtggtgggca cgacagcgaa gtgtcggggctgagcgtaga 1517 cctggcgaat agggtgatca gagcgcagtg cgcaggcatg cagccatacgtca 1570 2 376 PRT Corynebacterium glutamicum 2 Met Asn Thr Arg Val LeuGlu Ala Leu Pro Val Asp Leu Ala Asp Pro 1 5 10 15 Thr Ala Ile Leu GlyAsp Leu Glu Asp Ala Ile Ser Gly Lys Lys Thr 20 25 30 Phe Leu Pro Ile ProVal Gln Asp Lys Thr Arg Ala Gln Leu Leu Arg 35 40 45 Asp Ser Gln Arg ValGly Leu Ala Ile Asp Pro Ser Ile Ala Leu Val 50 55 60 Met Ala Thr Ser GlySer Thr Gly Thr Pro Lys Gly Ala Gln Leu Thr 65 70 75 80 Pro Leu Asn LeuVal Ser Ser Ala Asp Ala Thr His Gln Phe Leu Gly 85 90 95 Gly Glu Gly GlnTrp Leu Leu Ala Met Pro Ala His His Ile Ala Gly 100 105 110 Met Gln ValLeu Leu Arg Ser Leu Ile Ala Gly Val Glu Pro Leu Ala 115 120 125 Ile AspLeu Ser Thr Gly Phe His Ile Asp Ala Phe Ala Gly Ala Ala 130 135 140 AlaGlu Leu Lys Asn Thr Gly Asp Arg Val Tyr Thr Ser Leu Thr Pro 145 150 155160 Met Gln Leu Leu Lys Ala Met Asp Ser Leu Gln Gly Ile Glu Ala Leu 165170 175 Lys Leu Phe Asp Val Ile Leu Val Gly Gly Ala Ala Leu Ser Lys Gln180 185 190 Ala Arg Ile Ser Ala Glu Gln Leu Asp Ile Asn Ile Val Thr ThrTyr 195 200 205 Gly Ser Ser Glu Thr Ser Gly Gly Cys Val Tyr Asp Gly LysPro Ile 210 215 220 Pro Gly Ala Lys Val Arg Ile Ser Asp Glu Arg Ile GluLeu Gly Gly 225 230 235 240 Pro Met Ile Ala Gln Gly Tyr Arg Asn Ala ProGlu His Pro Asp Phe 245 250 255 Ala Asn Glu Gly Trp Phe Thr Thr Ser AspSer Gly Glu Leu His Asp 260 265 270 Gly Ile Leu Thr Val Thr Gly Arg ValAsp Thr Val Ile Asp Ser Gly 275 280 285 Gly Leu Lys Leu His Pro Glu ValLeu Glu Arg Ala Ile Ala Asp Ile 290 295 300 Lys Gly Val Thr Ala Ala CysVal Val Gly Ile Pro Asp Pro Arg Leu 305 310 315 320 Gly Gln Ala Ile ValAla Ala Tyr Ser Gly Ser Ile Ser Pro Ser Glu 325 330 335 Val Ile Glu GlyLeu Asp Asp Leu Pro Arg Trp Gln Leu Pro Lys Arg 340 345 350 Leu Lys HisLeu Glu Ser Leu Pro Ser Ile Gly Pro Gly Lys Ala Asp 355 360 365 Arg ArgAla Ile Ala Lys Leu Phe 370 375 3 19 DNA PCR primer 3 ctcactccgttgaatttgg 19 4 19 DNA PCR primer 4 caggtgcatt tctgtagcc 19

1. An isolated polynucleotide from coryneform bacteria, comprising apolynucleotide sequence which codes for the menE gene, chosen from thegroup consisting of a) polynucleotide which is identical to the extentof at least 70% to a polynucleotide which codes for a polypeptide whichcomprises the amino acid sequence of SEQ ID No. 2, b) polynucleotidewhich codes for a polypeptide which comprises an amino acid sequencewhich is identical to the extent of at least 70% to the amino acidsequence of SEQ ID No. 2, c) polynucleotide which is complementary tothe polynucleotides of a) or b), and d) polynucleotide comprising atleast 15 successive nucleotides of the polynucleotide sequence of a), b)or c), the polypeptide preferably having the activity ofO-succinylbenzoic acid CoA ligase.
 2. A polynucleotide as claimed inclaim 1, wherein the polynucleotide is a preferably recombinant DNAwhich is capable of replication in coryneform bacteria.
 3. Apolynucleotide as claimed in claim 1, wherein the polynucleotide is anRNA.
 4. A polynucleotide as claimed in claim 2, comprising the nucleicacid sequence as shown in SEQ ID No.
 1. 5. A DNA as claimed in claim 2which is capable of replication, comprising (i) the nucleotide sequenceshown in SEQ ID No. 1, or (ii) at least one sequence which correspondsto sequence (i) within the range of the degeneration of the geneticcode, or (iii) at least one sequence which hybridizes with the sequencecomplementary to sequence (i) or (ii), and optionally (iv) sensemutations of neutral function in (i).
 6. A DNA as claimed in claim 5which is capable of replication, wherein the hybridization is carriedout under a stringency corresponding to at most 2×SSC.
 7. Apolynucleotide sequence as claimed in claim 1, which codes for apolypeptide which comprises the amino acid sequences shown in SEQ ID No.2.
 8. A coryneform bacterium in which the menE gene is attenuated, inparticular eliminated.
 9. The integration vector pCR2.1menEint, which9.1. carries an internal fragment of the menE gene 520 bp in size, 9.2.the restriction map of which is reproduced in FIG. 1, and 9.3. which isdeposited in the E. coli strain Top10/pCR2.lmenEint under no. DSM 14080at the Deutsche Sammlung für Mikroorganismen und Zellenkulturen].
 10. Aprocess for the fermentative preparation of L-amino acids, in particularL-lysine, which comprises carrying out the following steps: a)fermentation of the coryneform bacteria which produce the desiredL-amino acid and in which at least the menE gene or nucleotide sequenceswhich code for it are attenuated, in particular eliminated; b)concentration of the L-amino acid in the medium or in the cells of thebacteria, and c) isolation of the L-amino acid.
 11. A process as claimedin claim 10, wherein bacteria in which further genes of the biosynthesispathway of the desired L-amino acid are additionally enhanced areemployed.
 12. A process as claimed in claim 10, wherein bacteria inwhich the metabolic pathways which reduce the formation of the desiredL-amino acid are at least partly eliminated are employed.
 13. A processas claimed in claim 10, wherein the expression of the polynucleotide(s)which code(s) for the menE gene is attenuated, in particular eliminated.14. A process as claimed in claim 10, wherein the catalytic propertiesof the polypeptide (enzyme protein) for which the polynucleotide menEcodes are reduced.
 15. A process as claimed in claim 10, wherein for thepreparation of L-amino acids, coryneform microorganisms in which at thesame time one or more of the genes chosen from the group consisting of15.1 the dapA gene which codes for dihydrodipicolinate synthase, 15.2the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase,15.3 the tpi gene which codes for triose phosphate isomerase, 15.4 thepgk gene which codes for 3-phosphoglycerate kinase, 15.5 the zwf genewhich codes for glucose 6-phosphate dehydrogenase, 15.6 the pyc genewhich codes for pyruvate carboxylase, 15.7 the mqo gene which codes formalate-quinone oxidoreductase, 15.8 the lysC gene which codes for afeed-back resistant aspartate kinase, 15.9 the lysE gene which codes forlysine export, 15.10 the hom gene which codes for homoserinedehydrogenase 15.11 the ilvA gene which codes for threonine dehydrataseor the ilvA(Fbr) allele which codes for a feed back resistant threoninedehydratase, 15.12 the ilvBN gene which codes for acetohydroxy-acidsynthase, 15.13 the ilvD gene which codes for dihydroxy-aciddehydratase, and 15.14 the zwal gene which codes for the Zwal protein isor are enhanced or over-expressed are fermented.
 16. A process asclaimed in claim 10, wherein for the preparation of L-amino acids,coryneform microorganisms in which at the same time one or more of thegenes chosen from the group consisting of 16.1 the pck gene which codesfor phosphoenol pyruvate carboxykinase, 16.2 the pgi gene which codesfor glucose 6-phosphate isomerase, 16.3 the poxB gene which codes forpyruvate oxidase, and 16.4 the zwa2 gene which codes for the Zwa2protein is or are attenuated are fermented.
 17. A coryneform bacteriumwhich contains a vector which carries parts of the polynucleotide asclaimed in claim 1, but at least 15 successive nucleotides of thesequence claimed.
 18. A process as claimed in one or more of thepreceding claims, wherein microorganisms of the species Corynebacteriumglutamicum are employed.
 19. A process for discovering RNA, cDNA and DNAin order to isolate nucleic acids, or polynucleotides or genes whichcode for O-succinylbenzoic acid CoA ligase or have a high similaritywith the sequence of the menE gene, which comprises employing thepolynucleotide comprising the polynucleotide sequences as claimed inclaims 1, 2, 3 or 4 as hybridization probes.